Comparative Study of Small-RNA and Degradome Sequencing Reveals Role of Novel stu-miR8006 in Regulating Root Development in Solanum tuberosum L.
Abstract
:1. Introduction
2. Materials and Methods
2.1. Plant Materials
2.2. Small-RNA Isolation and Sequencing
2.3. Sequencing Data Analysis and Identification of Potato miRNAs
2.4. Differential Expression Analysis of Potato miRNAs
2.5. Degradome Library Construction and Sequencing
2.6. Identification of miRNA Targets
2.7. RLM-5′-RACE Assay
2.8. qRT-PCR Analysis
2.9. Identification and Expressional Analysis of stu-miR8006-p5-1ss9AT
2.10. Vector Construction and Potato Transformation
2.11. Root Architecture Analysis
2.12. Statistical Analysis
3. Results
3.1. Overview of sRNA Sequencing
3.2. Identification of Known and Novel miRNAs in Potato
3.3. Differential Expression Analysis of miRNAs between ER and MR
3.4. Identification of miRNA Targets by Degradome Analysis
3.5. GO Enrichment and KEGG Pathway Analyses of Target Genes
3.6. Identification of miRNAs and Their Targets Involved in the Development of Potato Roots
3.7. Validation of miRNAs’ Targets Involved in the Development of Potato Roots
3.8. Expression Analysis of Root-Development-Related miRNAs and Their Target Genes
3.9. Clone, Characterization, and Expressional Analysis of stu-miR8006-p5-1ss9AT in Potato
3.10. Suppression of stu-miR8006-p5-1ss9AT Resulted in Alteration in Potato Root Architecture
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Data Availability Statement
Conflicts of Interest
References
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MiRNA | MiRNA Sequences | Target Gene | Transcript Annotation | GO_Term |
---|---|---|---|---|
PC-3p-230042_5 | CAAGGCTAGGACAGATGGAAAGAA | PGSC0003DMT400074930 | uncharacterized protein LOC102590046 isoform X1 | GO:0048364 (root development); GO: 0071433 (cell wall repair) |
PC-5p119431_10 | TCTTAGGCCTAATTCACACCC | PGSC0003DMT400003464 | receptor-like protein kinase FERONIA isoform X2 | GO:0030308 (negative regulation of cell growth); GO: 0048364 (root development) |
stu-miR477b-5p | ACTCTCCCTCAAAGGCTTCTG | PGSC0003DMT400021612 | serine/threonine-protein kinase CTR1-like isoform X1 | GO:2000069 (regulation of post-embryonic root development) |
stu-miR5303g | TTTTTGAAGAGTCTGGGCAAC | PGSC0003DMT400009997 | growth-regulating factor 9-like | GO:0008285 (negative regulation of cell proliferation); GO: 0048364 (root development) |
PGSC0003DMT400079970 | uncharacterized protein OsI_027940-like | GO:0010449 (root meristem growth); GO:0080037 (negative regulation of cytokinin-activated signaling pathway); GO:2000012 (regulation of auxin polar transport) | ||
stu-miR5303j-p5 | ATTCAAATTTCTGCCCTA | PGSC0003DMT400034518 | tropinone reductase-like 3 | GO:0048767 (root hair elongation) |
stu-miR8006-p5-1ss9AT | CATACTTTTGGGACATTG | PGSC0003DMT400013837 | auxin-induced in root cultures protein 12 isoform X1 | Function unknown |
PGSC0003DMT400040282 | protein NRT1/PTR FAMILY 1.2-like isoform X2 | GO:0010015 (root morphogenesis); |
Gene | Sequence of Oligonucleotides Forward (F) and Reverse (R) | Length (bp) |
---|---|---|
stu-miR8020 | R 5’-GGAATTTCATTGAGTATGTTGTTGTT-3’ | - |
stu-miR167d-3p | R 5’-CAGATCATGTGGTTGCTTCAC-3’ | - |
stu-miR477a-5p | R 5’-CCTCTCCCTCAAGGGCTTCT-3’ | - |
stu-miR408a-3p | R 5’-TGCACAGCCTCTTCCCTG-3’ | - |
stu-miR398a-3p | R 5’-TATGTTCTCAGGTCGCCCCT-3’ | - |
stu-MIR319-p5 | R 5’-TGCTGCTGAATCATTGGTTC-3’ | - |
PC-3p-28523_59 | R 5’-GCGTTATTTTCACCTGACAAGACT-3’ | - |
PC-5p-82220_15 | R 5’-GCGTTCTAGGATATAAATTGCACTACT-3’ | - |
PC-5p-138631_8 | R 5’-TCAACTATCCAGTCTTTCCTCAGA-3’ | - |
PC-5p-107179_11 | R 5’-CGAGTTTCCTACCTGAACTATCACC-3’ | - |
PC-5p-656396_2 | R 5’-ACTTCCTGATCACATGGCGA-3’ | - |
PC-3p-291253_4 | R 5’-TAAGGTAGGGCGCTCTTCG-3’ | - |
PC-3p-230042_5 | R 5’-CAAGGCTAGGACAGATGGAAAG-3’ | - |
PC-5p119431_10 | R 5’-GTCTTAGGCCTAATTCACACCC-3’ | - |
stu-miR477b-5p | R 5’-ACTCTCCCTCAAAGGCTTCTG-3’ | - |
stu-miR5303g | R 5’-GTTTTTGAAGAGTCTGGGCAAC-3’ | - |
stu-MIR5303j-p5 | R 5’-GCGATTCAAATTTCTGCCCTA-3’ | - |
stu-miR8006-p5-1ss9AT | R 5’-CGCATACTTTTGGGACATTG-3’ | - |
St18sRNA | R 5’-TTAGAGGAAGGAGAAGTCGTAACAA-3’ | - |
uncharacterized protein LOC102590046 | F 5’-CAAGCATACCCACTTCCTCT-3’ | 182 |
R 5’-GTCCTAACTCCTGAGACAACG-3’ | ||
receptor-like protein kinase FERONIA | F 5’-CGCCGAGGATAGTTTACACAA-3’ | 150 |
R 5’-ACCCTGTCGTTCACTTCCAG-3’ | ||
serine/threonine-protein ki’-se CTR1-like | F 5’-TTGTGTTACCGACGCAGT-3’ | 187 |
R 5’-GCAGGTTGCTTCAGTAGAGAT-3’ | ||
growth-regulating factor 9-like | F 5’-CAGAGCCAAGAAGATGTAGGAG-3’ | 168 |
R 5’-TTTGAAGAGTCCGAACAACG-3’ | ||
uncharacterized protein OsI_027940 | F 5’-TGACTTCTCGGACTGGCTGT-3’ | 182 |
R 5’-GCAGGATGTCTTCAACTCTGG-3’ | ||
tropinone reductase-like 3 | F 5’-CTGAAAGGCTTGGTTTAGAAGG-3’ | 225 |
R 5’-GCCACGGTAGGATTTACAGC-3’ | ||
auxin-induced in root cultures protein | F 5’-CGTATTCCCTTCCATCCTATC-3’ | 254 |
R 5’-GGCTGAGAAAGTTTGAGAGG-3’ | ||
protein NRT1/PTR FAMILY 1.2 | F 5’- CTGATTCTTACTGTGGTCGCT -3’ | 118 |
R 5’-CCTCTTGTTCTCGGGTTTC-3’ | ||
Stef1α | F 5’- ATTGGAAACGGATATGCTCCA-3’ | 101 |
R 5’- TCCTTACCTGAACGCCTGTCA-3’ |
Term | ER | MR |
---|---|---|
Raw reads | 15,443,787 | 12,099,703 |
3ADT and length filter | 2,202,430 (54.26%) | 6,595,079 (54.51%) |
Junk reads | 56,274 (0.36%) | 25,332 (0.21%) |
Rfam | 1,693,395 (10.96%) | 1,189,792 (9.83%) |
Repeats | 24,663 (0.16%) | 24,286 (0.20%) |
Valid reads | 8,985,433 (5%) | 3,166,534 (26.17%) |
Mapped reads | 46,382,473 (88.29%) | 44,040,313 (88.39%) |
Unique Mapped reads | 30,578,981 (58.21%) | 28,408,840 (57.02%) |
Q20 (%) | 99.73 | 99.72 |
Q30 (%) | 98.10 | 98.08 |
GC content (%) | 43 | 43.5 |
Type | Number of Reads | Percentage (%) |
---|---|---|
Raw reads | 26,207,517 | 100 |
Reads < 15 nt after removing 3 adapters | 229,844 | 0.88 |
Mappable reads | 25,977,673 | 99.12 |
Unique raw reads | 9,690,091 | 100 |
Unique reads < 15 nt after removing 3 adapters | 78,098 | 0.81 |
Unique mappable reads | 9,611,993 | 99.19 |
Transcript mapped reads | 18,191,670 | 69.41 |
Unique transcript mapped reads | 5,637,124 | 58.17 |
Number of input transcript | 56,218 | 100 |
Number of covered transcript | 43,889 | 78.07 |
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Duan, X.; Yang, J.; Zhang, F.; Han, Y.; Gong, Y.; Liu, M.; Zhang, N.; Si, H. Comparative Study of Small-RNA and Degradome Sequencing Reveals Role of Novel stu-miR8006 in Regulating Root Development in Solanum tuberosum L. Agronomy 2023, 13, 2942. https://doi.org/10.3390/agronomy13122942
Duan X, Yang J, Zhang F, Han Y, Gong Y, Liu M, Zhang N, Si H. Comparative Study of Small-RNA and Degradome Sequencing Reveals Role of Novel stu-miR8006 in Regulating Root Development in Solanum tuberosum L. Agronomy. 2023; 13(12):2942. https://doi.org/10.3390/agronomy13122942
Chicago/Turabian StyleDuan, Xiaoqin, Jiangwei Yang, Feiyan Zhang, Yuwen Han, Yating Gong, Mei Liu, Ning Zhang, and Huaijun Si. 2023. "Comparative Study of Small-RNA and Degradome Sequencing Reveals Role of Novel stu-miR8006 in Regulating Root Development in Solanum tuberosum L." Agronomy 13, no. 12: 2942. https://doi.org/10.3390/agronomy13122942